The present invention relates to (poly)peptides that are recognized by anti-HHV-8 antibodies of HHV-8 infected patients. By definition, these (poly)peptides shall not comprise naturally occurring HHV-8 proteins. The invention further relates to polymers containing two or more (identical or different) inventory peptides as well as conjugates comprising the inventory peptides and/or polymers thereof. Further we describe mixtures comprising the inventory peptides and/or polymers thereof that are particularly suited for use in procedures to detect anti-HHV-8 antibodies with high sensitivity and specificity. In addition, the present invention relates to a diagnostic kit comprising the inventory peptides, polymers and/or mixtures thereof for the detection of anti-HHV-8 antibodies or diagnosis of HHV-8 infection, respectively.
The human herpesvirus 8 (HHV-8), also known as Kaposi sarcoma associated herpesvirus (KSHV) is the purported etiological agent of Kaposi""s sarcoma and certain B cell lymphomas. Kaposi""s sarcoma (KS) is the most frequent tumor in AIDS patients, affecting 20-30% of all patients during the course of their HIV infection. In the U.S., the incidence of KS in this risk group is 20,000fold higher than in the general population [1]. In some geographic areas, e.g. in Mediterranean countries or in Africa, the incidence in the general population is significantly higher.
AIDS-associated KS is characterized by an aggressive course and high mortality whereas classical KS is generally relatively indolent and slowly progressing. Other forms of the disease are endemic KS that predominantly affects children and adolescents in Sub-Saharan Africa, and iatrogenic KS in inmnune suppressed transplant recipients.
KS was first described more than a hundred years ago as a relatively rare tumor occurring predominantly in elderly men of Mediterranean or East European origin [2]. The characteristic external manifestations of KS are sharply defined, differently colored (purple, brown, violet or black) nodular lesions of the skin, mostly on the extremities but also in oral mucosa and viscera. Histologically, these lesions consist of long spindle-shaped cells, apparently of endothelial origin, as well as a number of other cell types like fibroblasts, neo-vascular structures, infiltrating leukocytes and extra-vasated red blood cells.
The production of VEGF (vascular endothelial growth factor) is significantly increased in tumor tissue leading to continued angiogenesis and, hence, an extreme vascularization. Already years ago, epidemiological analyses gave hints that an infectious agent might be involved in the development of KS [1, 3]. Using a new PCR technique, Chang and Moore isolated from KS biopsies DNAs from a hitherto unknown human xcex3-2 herpesvirus [4]. This virus was called KSHV (Kaposi sarcoma associated herpesvirus) or HHV-8 (human herpesvirus 8), respectively. Its 140 kb genome has been recently cloned and sequenced [5].
Since then a series of publications has shown beyond reasonable doubt that all forms of Kaposi""s sarcoma are correlated in practically 100% of all cases with the presence of HHV-8 in KS lesions [6-11]. Moreover, PCR detection of HHV-8 is a prognostic marker for later development of Kaposi""s sarcoma [12]. Our own work has shown that seroconversion to HHV-8 is detectable on the average already two years before the clinical manifestation of KS.
Most likely HHV-8 is also involved in the pathogenesis of certain lymphoproliferative disorders such as multicentric Castleman""s disease (MCD) or primary effusion lymphoma [13, 14]. Some data also hint to a contribution of HHV-8 in interstitial pneumonia and encephalitis [15]. Recently published work suggests that HHV-8 is correlated with multiple myeloma [16]. Although several experimental results are compatible with the hypothesis that HHV-8 may be involved in the pathogenesis of this disease [17-22], a causal relationship is not yet proven. In some geographic regions such as Central Africa, HHV-8 appears to be relatively widespread in the general population [8, 23-25]. Despite a higher incidence of KS in these countries, the presence of an infection with HHV-8 alone appears to be necessary but not sufficient for the development of this disease.
Based on the available viral DNA sequences, several diagnostic tests have been developed that allow a direct or indirect detection of HHV-8. With the help of a relatively simple test, the polymerase chain reaction, even minute quantities of viral DNA can be detected in blood or tissue samples [14, 26-28]. However, for some medical analyses PCR tests are not sensitive enough.
Immunosuppressed organ transplant recipients also have an increased risk of developing Kaposi""s sarcoma (up to 5% for kidney transplant recipients); the number of cases varies greatly with geographic origin. In countries of the Near and Middle East, KS is the most common post transplant tumor; worldwide it ranks third. In developed countries (Europe, U.S. or Japan) about 1 to 1.5 m people have an increased risk of getting KS. Blood products for these patients should therefore be unconditionally tested for anti-HHV-8 antibodies as a marker for a possible contamination with HHV-8 in order to keep their infection risk as small as possible. In a broader sense this also applies for other more or less immunocompromised groups, e.g. patients under high dose chemotherapy, dialysis patients, elderly people, neonates etc.
KS predominantly affects people suffering from various forms of immunodeficiency, and is only poorly treatable by chemotherapy, surgery or with ionizing radiation. Presently, there is neither a causative treatment nor a final cure for this disease. One has to consider that immune deficient individuals are exposed to an extraordinary risk to develop Kaposi""s sarcoma if they receive HHV-8 contaminated blood products.
For routine diagnostics purposes one mostly uses ELISAs (enzyme-linked immunosorbent assays) because these tests are cheap and suitable for high throughput testing. The detection of HHV-8 specific antibodies indicates prior contact with HHV-8 antigens. This antibody detection can be positive even when the amount of viral DNA (in blood) is below the level of detection in a PCR analysis. Except for ELISAs [29-31], the presence of HHV-8 specific antibodies can also be detected by indirect immunofluorescence assays (IFAs) [25] or Western blots [32, 33].
However, the test procedures that have been described so far are either very time consuming (IFA or Western blot) or not sensitive enough (PCR from blood samples, ELISAs using previously described antigens.
The underlying technical problem for this invention was therefore to define reagents the use of which allows a very sensitive and specific detection of anti-HHV-8 antibodies.
This technical problem is solved by presenting the embodiments detailed in the claims section.
Thus the present invention relates to a (poly)peptide, which is recognized by anti-HHV-8 antibodies in HHV-8 infected patients and is characterized as follows:
(a) it comprises one of the amino acid sequences presented in SEQ ID nos. 1 to 8;
(b) consists of one of the amino acid sequences presented in SEQ ID nos. 1 to 8 or
(c) consists of an amino acid sequence which differs from one of the amino acid sequences described in (a) or (b) by one or several substitutions, deletions and/or insertions; wherein the (poly)peptide is not a naturally occurring HHV-8 protein.
Naturally occurring HHV-8 proteins in the sense of this invention are HHV-8 proteins that have the full-length amino acid sequence and are not degraded. Usually or preferentially, the (poly)peptide is recognized by anti-HHV-8 antibodies in bodily fluids of HHV-8 infected individuals. Methods for the determination of the antigenic property of the (poly)peptide related to the invention as well as methods for the isolation of anti-HHV-8 antibodies, preferably from bodily fluids of HHV-8 infected patients are known to those skilled in the art. Such methods comprise for example ELISA tests.
Bodily fluids in the sense of the present invention encompass e.g. blood, serum, plasma, lymph fluid, tissue fluid and extracts, e.g. from mucosa of respiratory, urogenital or gastro-intestinal origin. Further, this expression comprises bodily fluids that have been pretreated to be usable for the above mentioned analysis methods. Examples for this are sera diluted in vitro or treated with preservative agents for cryoprotection (e.g. glycerol) or coagulation inhibition (e.g. heparin).
The term xe2x80x9c(poly)peptidexe2x80x9d as used in relation to the present invention comprises both peptides and polypeptides. (Poly)peptides related to the invention, which comprise one or more of the amino acid sequences characterized in SEQ ID no. 1 to 8 can be flanked by another HHV-8 sequence as well as by a sequence unrelated to HHV-8. As already mentioned above, said (poly)peptide is not a naturally occurring HHV-8 protein, i.e. said (poly)peptide is not a full length HHV-8 protein as coded by HHV-8 genomes.
Preferably, said (poly)peptides are linear epitopes of different coding regions of the HHV-8 genome, and are less than 50 amino acids in length. More preferred is a length of maximal 15 amino acids, and most preferred is a length of the (poly)peptides related to the invention of 10 to 12 amino acids.
The term xe2x80x9csubstitutionxe2x80x9d in the sense of the present invention comprises both conservative and non-conservative exchanges of amino acids. Conservative exchanges are those in which a neutral, hydrophobic, polar, basic or acidic amino acid is replaced by an amino acid of the same class. The different classes of amino acids, the classification of which is determined by specific side chains is well known to those skilled in the art. The crucial point is that the three-dimensional structure of the (poly)peptides is not perturbed in such a way that the peptide is no longer recognized by anti-HHV-8 antibodies.
The peptides related to the invention were identified in a screening of about 3000 peptides according to the following criteria:
(a) minimal homology to peptides of known proteins, in particular to corresponding epitopes of related herpesviruses as for instance EBV and CMV, and
(b) maximal antigenicity (by computer prediction)
A preselection of potentially immunodominant HHV-8 specific peptide sequences was achieved by an advantageous combination of different computer programs (BLASTP, ANTIGENIC and BLASTALIGN) of the Genetics Computer Group (GCG), Wisconsin.
All peptides selected by this procedure (292) were then individually tested with a serum pool from KS patients and several control pools from healthy individuals.
The peptides that had passed this initial screen were then analyzed with several hundred sera from KS patients and controls with special attention given to cross-reactions with EBV-, CMV- or HSV-specific antibodies. By these multiple tests, the peptides according to the invention were identified that showed the desired reaction profile.
The (poly)peptides also comprise compounds that are produced by peptidomimetics, preferably (poly)peptides resembling the above mentioned (poly)peptides in their immunological and diagnostic properties. It is known in the art how such molecules can be designed and produced using L- and D-amino acids (e.g. Banerjee, Biopolymers 39 (1996), 769-777). Preferably, the present invention relates to a peptide mix consisting of a combination of said (poly)peptides.
In a preferred embodiment, the peptide mix comprises the (poly)peptides disclosed in SEQ ID nos. 1, 3, 4 and 8.
In an especially preferred embodiment the peptide mix related to the invention contains these (poly)peptides in a molar ratio of 1:1:1:1.
Using the peptide mix according to the invention, anti-HHV-8 antibodies can be detected in an especially advantageous way. Surprisingly, the peptide mix surpasses by far both the sensitivity and the specificity of traditional detection methods (see below).
In another preferred embodiment, the (poly)peptide comprising an amino acid sequence that differs from the amino acid sequences shown in (a) by one or more substitutions, deletions and/or insertions is a naturally occurring sequence variant.
Such peptides that represent sequence variants of the corresponding positions in the genome of HHV-8 (5) may further increase the sensitivity of the detection. The specificity remains unaltered as the selection of peptides by minimal homology to known (viral) proteins excludes highly conserved regions to start with.
In another embodiment, the present invention concerns a polymer comprising at least two identical or different said (poly)peptides .
In the sense of the present invention, such polymers comprise homopolymers consisting of several copies of a single (poly)peptide as well as heteropolymers of any combination of said (poly)peptides whereby such heteropolymers may also contain several copies of one said (poly)peptide.
In a preferred embodiment, the polymer related to the invention is characterized by an unbranched chain of polymerized said (poly)peptides.
In another preferred embodiment, said (poly)peptide is characterized by branched chains of polymerized said (poly)peptides.
Polymers with branched chains of polymerized (poly)peptides are produced by connecting peptides via an amino acid or an amino acid analog that possess two amino groups and one carboxyl group each capable of forming peptide bonds. Such procedures are known in the art.
In another preferred embodiment, the present invention concerns polymers that comprise peptides with the amino acid sequences disclosed in SEQ ID nos. 1, 3, 4 and 8.
In another preferred embodiment, said (poly)peptide is chemically synthesized.
Procedures for chemical synthesis of peptides using peptide synthesizers are known in the art. Preferably, the present invention relates to mixtures consisting of the polymers according to the invention.
In another embodiment, the present invention relates to a fusion protein comprising a (poly)peptide or polymer according to the invention.
In another embodiment, the present invention concerns a polynucleotide coding for a (poly)-peptide, polymer or fusion protein according to the invention.
In a preferred embodiment, the polynucleotide is DNA or RNA.
The present invention further relates to a vector comprising a polynucleotide according to the invention.
In a preferred embodiment, the vector according to the invention is an expression vector.
The term xe2x80x9cexpression vectorxe2x80x9d in the sense of the present invention comprises both prokaryotic and eukaryotic expression vectors. The necessary regulatory elements for the expression of a (poly)peptide are known in the art and can be selected to achieve the desired expression. The term xe2x80x9cexpressionxe2x80x9d means transcription as well as transcription and translation. In particular, regulatory elements comprise promoters. For expression in prokaryotic cells of a poly-nucleotide according to the invention a series of suitable promoters exist, e.g. the E. coli lac or trp promoter, the lambda phage PR- or PL-promoter lacI, lacZ, T3, T7, gpt etc. Eukaryotic promoters include for instance the CMV immediate early promoter, the HSV promoter, the thymidine kinase promoter, the SV40 promoter, LTRs of retroviruses or the mouse metallothioneinl promoter. A multitude of expression vectors has been described for expression in prokaryotic as well as eukaryotic cells, e.g. for eukaryotes pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) or GEM1 (Promega Biotech, Madison, Wis., USA), pSV2CAT, pOG44 and for prokaryotes pQE70, pQE60, pBluescript SK, etc. In addition to promoters, vectors according to the invention may also contain other elements that further increase the transcription, e.g. so-called enhancers. Examples are the SV40 enhancer, the polyoma enhancer, the cytomegalovirus early promoter-enhancer and the adenovirus enhancer.
In another embodiment, the present invention relates to a host cell containing an expression vector according to the invention.
The term xe2x80x9chost cellxe2x80x9d according to the invention comprises prokaryotic as well as eukaryotic host cells. Preferred prokaryotic host cells include e.g. E. coli cells, Streptomyces, Bacillus or Salmonella cells, preferred eukaryotic host cells include yeast cells, in particular Saccharomyces cerevisiae cells, insect cells as e.g. Drosophila or SF9 cells, animal cells as e.g. CHO or COS cells, plant cells or mammalian cells.
In another embodiment, the present invention relates to a procedure for production of a (poly)peptide, polymer or fusion protein according to the invention comprising the following steps:
(a) cultivation of a host cell according to the invention under conditions that foster the expression of the (poly)peptide, polymer or fusion protein; or
(b) in vitro transcription and/or translation of the polynucleotide according to the invention;
and isolation of the (poly)peptide, polymer or fusion protein produced as under (a) or (b).
The conditions for expression of a (poly)peptide are known in the art and can be selected according to the host cell and the expression vector used. The same is true for the isolation of the expression product. When an expression vector is used that leads to the secretion of the synthesized (poly)peptide, the (poly)peptide is isolated from the culture supernatant. When expression takes place intracellularly, the expression product is isolated from the host cell. Methods for in vitro transcription and/or translation are well known in the art. For instance, such procedures may be performed using commercial kits according to the instructions of the manufacturer.
Preferably, additional purification steps known in the art, e.g. column chromatography, may be used for the isolation of said (poly)peptide by removing impurities such as cellular proteins, nucleic acids or components of the in vitro transcription/translation system.
In another embodiment, the present invention relates to a conjugate comprising a (poly)peptide and/or polymer and/or fusion protein according to the invention or a (poly)peptide and/or polymer and/or fusion protein that is produced by said procedure.
In another embodiment, the present invention concerns a composition comprising at least one (poly)peptide and/or polymer and/or fusion protein according to the invention and a conjugate according to the invention, where appropriate also a pharmaceutically compatible carrier and/or diluent.
In a preferred embodiment, the composition is a pharmaceutical.
In an especially preferred embodiment, the pharmaceutical is a vaccine. In this embodiment, the peptides are preferably coupled to a carrier, either individually or in combination. Examples for suitable carriers are polystyrene beads, streptavidin, BSA or KLH.
Examples for suitable pharmaceutically compatible carriers and/or diluents are known in the art and comprise e.g. phosphate buffered sodium chloride solutions, water, emulsions as for instance oilwater emulsions, various forms of surfactants and detergents, sterile solutions etc. Pharmaceuticals comprising such carriers can be formulated according to known conventional methods. These pharmaceuticals may be administered to an individual in a practical dosis, either orally or parenterally (e.g. intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, intranasally, intrabronchially or intradermally or through a catheter into an artery. The physician in charge according to clinical factors determines the dosage and mode of administration. It is known in the art that the dosage depends on various factors such as height, weight, body surface, age, sex or the general health of the patient but also on the characteristics of the administered pharmaceutical, the duration and mode of application as well as on other drugs that may be given simultaneously. A typical dosis may be in a range of 0.001 and 1000 mg whereby dosages below or above this range are conceivable, in particular in consideration of the above mentioned factors. With regular administration of the composition according to the invention, the dosage should generally fall in a range between 1 xcexcg and 10 mg units per day. In case of an intravenous administration (which is not recommended because of the danger of anaphylactic shock), the dosis should be in a range between 1 xcexcg and 10 mg per kg of body weight per minute.
The composition of the invention can be administered locally or systemically. Preparations for a parenteral administration comprise sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples for non-aqueous solvents are propylenglycol, poly-ethylenglycol, plant oils as e.g. olive oil and organic ester compounds as e.g. ethyloleate that are suitable for injections. Aqueous carriers include water, alcohol-water solutions, emulsions, suspensions, salt solutions and buffered media. Parenteral carriers include sodium chloride solutions, Ringer-dextrose, dextrose and sodium chloride, Ringer-lactate and bonded oils. Intravenous carriers include fluid, nutrient and electrolyte supplements (e.g. those based on Ringer-dextrose). The composition according to the invention may also contain preservatives and other supplements such as antimicrobial compounds, antioxidants, complexing agents and inert gases. Furthermore, depending on the intended use, the composition according to the invention may contain compounds such as interleukins, growth factors, differentiation factors, interferons, chemotactic proteins or unspecifically immunomodulatory agents.
In another embodiment, the present invention relates to a procedure for the detection of anti-HHV-8 antibodies comprising the following steps:
(a) contacting a biological sample with at least one of the (poly)peptides and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein as produced by said method , and/or a conjugate according to the invention, under conditions that allow the binding of antibodies; and
(b) detection of the antibodies bound in step (a)
In addition to the detection of anti-HHV-8 antibodies, said procedure is useful as prognostic marker for the eventual development of Kaposi""s sarcoma in HIV infected individuals or persons with other natural or iatrogenic immune defects.
In another preferred embodiment, this procedure uses a mixture of (poly)peptides as characterized by the amino acid sequences disclosed in SEQ ID nos. 1, 3, 4 and 8.
The main advantages of said method with respect to previously described procedures [25, 29-34] are on one hand the high sensitivity ( greater than 96% of sera from KS patients are positive as opposed to most procedures described in the literature, which only achieve sensitivities between 35 and 85%) and on the other hand a very good specificity and reproducibility (with the ELISA technique duplicate variations are generally less than 5%).
In particular, the use of a combination of different (poly)peptides from different open reading frames of the viral genome rather than individual recombinant antigens such as ORF65 [30] or the Minor Capsid Protein [31] allows the detection of a wider spectrum of HHV-8 specific antibodies. This is especially important with sera from patients who already suffered from a marked immunodeficiency when they were infected with HHV-8 because the antibody titers may be very low in these cases. Other peptide-based immunoassays that use a single peptide achieve a much lower sensitivity [29]. In addition, it is known from other herpesviruses that the use of a single epitope is not sufficient to obtain a diagnostically relevant recognition quota of at least 90% of the true positive sera.
In an especially preferred embodiment of this procedure, said (poly)peptides are chemically synthesized.
By using chemically synthesized peptides rather than recombinant antigens from prokaryotic expression systems or unpurified viral lysates, respectively as employed in other described procedures, a reaction with non-specific antibodies (e.g. against E. coli proteins or against proteins as e.g. EBV or CMV) is practically excluded. In hitherto described procedures, the antigens have to be present at a high degree of purity as even minute amounts of contaminating material (e.g. residual E. coli proteins) can lead to false positive results in many samples. A significant advantage of said procedure is that it does not require such time-consuming and costly purification steps (as e.g. prior adsorption of the test sera to E. coli lysates).
Preferably, the (poly)peptides and/or polymers and/or fusion proteins according to the invention are biotinylated for use in said procedure. The biotinylated components are then bound to a streptavidin-coated solid phase via the highly specific biotin-streptavidin interaction. This coupling procedure ensures an even coating even with mixtures of said (poly)peptides that are different in their charge or their hydrophobicity, respectively.
In another especially preferred embodiment of this procedure, the mixture contains these (poly)peptides in an equimolar ratio.
In another preferred embodiment, said procedure is an enzyme-linked immunosorbent assay (ELISA), an immunodot assay, an immunobead assay, a passive hemagglutination assay (PHA) or a peptide-antibody-peptide sandwich assay.
In another preferred embodiment of said procedure, the biological sample is a pretreated or untreated form of blood, serum, tissue extract, tissue fluid, cell culture supernatant or cell lysate. The term xe2x80x9cpretreated formxe2x80x9d as used in the sense of the present invention, the reader is redirected to the above definition of the term xe2x80x9cbodily fluidsxe2x80x9d since the possibilities of pretreatment and preparation are also applicable in this context.
In another preferred embodiment, said procedure comprises the additional step:
(c) detection of unspecific binding reactions in step (a) caused by other antibodies that may be present in the biological sample
In ELISA tests, additional antibodies may bind independently of the antigen or unspecifically to the surface of the wells of a microplate, thus causing a false positive signal. False positive results due to unspecific binding to the matrix can be safely detected and eliminated by using individual blank wells (without specific antigen). This is of importance for screening of sera in blood banks in order to avoid that donor blood is unnecessarily discarded due to false positive results of HHV-8 tests.
The present invention further relates to a kit, comprising:
(a) at least one (poly)peptide and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein produced according to the invention, and/or a conjugate according to the invention whereby the components may be separated or in form of a mixture; and where appropriate
(b) a solid phase to which at least one (poly)peptide and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein produced according to the invention, and/or a conjugate according to the invention may be bound;
(c) a sample diluent; and/or
(d) a negative control; and/or
(e) a positive control; and/or
(f) a reporter molecule
The term xe2x80x9creporter moleculexe2x80x9d in the sense of the present invention comprises enzymes as e.g. peroxidase or alkaline phosphatase (in these cases the detection system may be based on a colorimetric reaction as the conversion of ABTS by a peroxidase) as well as other enzymes, radioisotopes, fluorophores, bioluminescent or chemiluminescent molecules and the like or conjugates with e.g. a secondary antibody recognizing e.g. human IgG or IgM antibodies.
Said kit can be used to detect anti-HHV-8 antibodies and to diagnose HHV-8 infections of mammals, preferably in bodily fluids such as blood, serum, tissue extracts, tissue fluids, as well as in in vitro cell culture supernatants and cell lysates (as test reagents). For the intended use, any suitable immunoassay may be chosen, in particular an enzyme-linked immunosorbent assay (ELISA), an immunodot assay, an immunobead assay, a passive hemagglutination assay (PHA), a peptide-antibody-peptide sandwich assay or other methods known in the art.
In a preferred embodiment, the kit contains a mixture of the (poly)peptides consisting of or containing the amino acid sequences disclosed in SEQ ID nos. 1, 3, 4 and 8.
In another embodiment, the present invention relates to the use of at least one (poly)peptide and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein produced according to the invention for the production of a pharmaceutical for treatment of HHV-8 associated diseases.
In a preferred embodiment of the use according to the invention, the HHV-8 associated diseases are Kaposi""s sarcoma, multicentric Castleman""s disease, primary effusion lymphoma, interstitial pneumonitis, encephalitis and multiple myeloma.
In another embodiment, the present invention concerns the use of at least one (poly)peptide and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein produced according to the invention, and/or conjugate according to the invention for the production of antibodies.
The production of antibodies is well known in the art and comprises e.g. the immunization of an animal with an antigen whereby the antigen may be coupled to a carrier and/or given in combination with other immunostimulating substances (cf. e.g. Harlow and Lane, xe2x80x9cAntibodies, a laboratory manualxe2x80x9d, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988). Other known methods include the screening of antibody libraries with the xe2x80x9cphage displayxe2x80x9d technology and the recombinant production of the desired antibody.
In another embodiment the present invention concerns the use of at least one (poly)peptide and/or polymer and/or fusion protein according to the invention, a (poly)peptide and/or polymer and/or fusion protein produced according to the invention, and/or conjugate according to the invention for the detection of anti-HHV-8 antibodies.
The references cited in the present description are herewith defined to be part of the description.